Cpt1 α

Mouse tissue was homogenized in RIPA assay buffer, and the protein content was determined using a bicinchoninic acid protein assay kit (Pierce, IL, USA). The following antibodies were used: apolipoprotein B (ApoB) and GAPDH (Millipore, MA, USA); AKT, phospho-AKT (Ser473) (Cell Signaling Technology, MA, USA); PPARα (GeneTex, CA, USA); SCD1 (BioVision, CA, USA); IRS1 (Santa Cruz Biotechnology, CA, USA); CPT1α (Proteintech, IL, USA). The protein bands were analyzed using densitometry and Image J image analysis software. The exam proteins levels were normalized to that of GAPDH. The results were represented by the ratio of the values to WT NC group.

Dapagliflozin was obtained from Apexbio (United States, ^#A5854), with a purity of ≥99%; TGF-β1was obtained from PeproTech (United States). The western blot antibodies used were Collagen 1 (Abcam, United States, ^#ab270993), α-SMA (Sigma, United States, ^#A5691), vimentin (Proteintech, United States, ^#10366-1-AP), VDAC1(Proteintech, United States, ^#55259-1-AP), FN (Proteintech, United States, ^#15613-1-AP), p-JNK (CST, United States, ^#4668), JNK (CST, United States, ^#9252), p-ERK (CST, United States, ^#4370), ERK (CST, United States, ^#4695), P-P38 (CST, United States, ^#4631), p38 (CST, United States, ^#8690), total oxphos complexes (Thermo, United States, AB-2533835), CPT1-α (Proteintech, United States, ^#15184-1-AP), 0.2% adenine diet (TP 1S002), and control diet (LAD 3001) obtained from Trophic Animal Feed High-tech Co., Ltd., China. The SOD activity kit (^#A001-3), GSH content assay kit (^#A006-2-1) and MDA content assay kit (^#A003-1) were purchased from the Nanjing Jiancheng Institute of Bioengineering.

Liver tissue samples and cultured cells were lysed in RIPA buffer. The nuclear and cytoplasmic protein fractions were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Protein concentration was measured using a BCA protein assay kit (Boster, Wuhan, China). The protein samples were separated by SDS–PAGE (Bio–Rad Laboratories Inc., Berkeley, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp, Billerica, MA, USA). The PVDF membranes were incubated with primary antibodies against PGC-1α, BNIP3, Beclin-1, MCAD, CPT-1α (1:500; Proteintech, Wuhan, China), PPAR-α (1:500; Cell Signaling Technology, USA), Hif-2α and LC3B (1:1000; Abcam, Cambridge, MA, USA), and Lamin A/C (1:500; Zhongshan Jinqiao, China) for 24 h, and then incubated with secondary antibody (1:500; Zhongshan Jinqiao, China) for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (ECL) assay (Thermo Scientific, USA), and the blots were analyzed using Image Lab 3.0 (Bio–Rad, Hercules, CA, USA). The gray value of protein bands was calculated using Image J 1.50i (National Institutes of Health, USA) and normalized to that of the internal control, β-actin.